Keywords
Extracellular vesicles, Biomarkers, Lipidomic, Proteomics
Background
Multiple sclerosis (MS) is a chronic demyelinating disease of the CNS with no clear disease specific biomarkers or reliable markers to distinguish MS subtypes. Extracellular Vesicles (EVs) carry cell-specific proteins and lipids and cross the blood–brain barrier, making them a promising non-invasive indicator of disease activity in MS.
Objective
To identify distinct proteomic and lipidomic signatures of plasma-derived EVs in Relapsing-Remitting MS (RRMS) and Progressive MS (PMS) compared to Healthy Controls (HCs), and to evaluate whether these signatures correlate with clinical features, supporting their potential as peripheral biomarkers for disease as well as subtype differentiation.
Methods
EVs were isolated from 500μL of plasma using Size Exclusion Chromatography optimised for purity and yield. Targeted lipidomic (177 lipid targets) was performed on a pilot cohort of RRMS (n=10), PMS (n=8), and HCs (n=10). Proteomics was performed on RRMS (n=43), PMS (n=21), and HCs (n=43). EVs were characterised by nanoparticle tracking analysis using Zetaview and transmission electron microscopy. High-resolution LC-MS/MS using Data-Independent-Acquisition (DIA) in Lumos (Orbitrap) was used for proteomic analysis and C18 chromatography using a Vanquish HPLC coupled to a Thermo Altis TSQ triple quadrupole mass spectrometer was used for targeted lipidomic analysis of EVs and matched plasma.
Results
Lipidomic analysis revealed elevated EV-associated Lysophosphatidylethanolamine (18:0), Phosphatidylinositol (18:0/20:4), Lactosylceramide (18:1/22:0), and reduced Acylcarnitine (6:0) in MS versus HCs. No significant changes were found in matched plasma. Proteomic profiling identified emerging EV-associated markers, including increased T-complex protein-1 subunit delta and decreased Immunoglobulin kappa variable 3D-15 in RRMS, and increased Peroxiredoxin-1 with reduced immunoglobulin chains in PMS compared to HCs. These EV-specific changes indicate distinct disease signatures not observable in plasma alone.
Conclusion
This is the first study to simultaneously profile lipids and proteins in plasma-derived EVs and matched plasma in MS. Early findings reveal disease-specific EV lipid signatures and subtype-specific protein differences, supporting further validation in larger cohorts and integration with clinical data for improved subtype discrimination.